Leveraging TCR Affinity in Adoptive Immunotherapy against Shared Tumor/Self-Antigens

Abstract

Adoptive cellular therapy (ACT) using T-cell receptor (TCR)-engineered lymphocytes holds promise for eradication of disseminated tumors but also an inherent risk of pathologic autoimmunity if targeted antigens or antigenic mimics are expressed by normal tissues. We evaluated whether modulating TCR affinity could allow CD8⁺ T cells to control tumor outgrowth without inducing concomitant autoimmunity in a preclinical murine model of ACT. RIP-mOVA mice express a membrane-bound form of chicken ovalbumin (mOVA) as a self-antigen in kidney and pancreas. Such mice were implanted with OVA-expressing ID8 ovarian carcinoma cells and subsequently treated with CD8⁺ T lymphocytes (CTL) expressing either a high-affinity (OT-I) or low-affinity (OT-3) OVA-specific TCR. The effects on tumor growth versus organ-specific autoimmunity were subsequently monitored. High-affinity OT-I CTLs underwent activation and proliferation in both tumor-draining and pancreatic lymph nodes, leading to both rapid eradication of ID8-OVA tumors and autoimmune diabetes in all treated mice. Remarkably, the low-affinity OT-3 T cells were activated only by tumor-derived antigen and mediated transient regression of ID8-OVA tumors without concomitant autoimmunity. The OT-3 cells eventually upregulated inhibitory receptors PD-1, TIM-3, and LAG-3 and became functionally unresponsive, however, allowing the tumors in treated mice to reestablish progressive growth. Antibody-mediated blockade of the inhibitory receptors prevented exhaustion and allowed tumor clearance, but these mice also developed autoimmune diabetes. The findings reveal that low-affinity TCRs can mediate tumor regression and that functional avidity can discriminate between tumor-derived and endogenous antigen, while highlighting the risks involved in immune-checkpoint blockade on endogenous self-reactive T cells.

Fig. 1.. Induction of autoimmune diabetes by OT-3 cytotoxic T lymphocytes.

A) Systemic activation of 10⁶ adoptively transferred OT-3 CD8⁺ T cells by intravenous (i.v.) cotransfer of 3,000 CFU of OVA-expressing Listeria monocytogenes (Lm-OVA) resulted in autoimmune destruction of beta islets in RIP-mOVA mice. B) Systemic activation of OT-3 CD8 T cells by intravenous (i.v.) cotransfer of 10⁶ CFU of Lm-OVA deficient for Act A (Lm-OVA Act A−/−) is sufficient to result in autoimmune diabetes. C) Autoimmune diabetes induced by adoptively transferred OT-3 CD8⁺ T cotransferred with 3,000 CFU of Lm-OVA cells is dose-dependent, requiring 10⁵ OT-3 T cells to develop diabetes at 100% penetrance of the phenotype.

Fig. 2.. ID8-OVA tumors induce proliferation of adoptively transferred OT-3 CD8 T cells by cross presentation.

A) RIP-mOVA mice bearing 3-week old ID8-OVA tumors elicited proliferation of adoptively transferred OT-3 CD8⁺ T cells that were identifiable by the congenic marker CD45.1 and labeled with cell-trace violet (CTV) within the tumor draining lymph node 72 hours post-transfer, whereas mice lacking tumors did not demonstrate proliferation. B) Adoptively transferred OT-3 CD8⁺ T cells into tumor bearing mice produced Granzyme-B 72 hours post-transfer, consistent with functional activation.

Fig. 3.. OT-1 and OT-3 CD8 T cells have different thresholds of anti-tumor reactivity versus autoimmune diabetes.

A) Adoptive cell transfer of 5×10⁶ naïve OT-I CD8⁺ T cells, prepared by negative selection from donor transgenic OT-I mice splenocytes and lymph nodes, mediates destruction of ID8-OVA tumors as reflected by decreased tumor bioluminescence. However, all recipient mice develop autoimmune diabetes. ACT of OT-3 CD8⁺ T cells, in contrast, mediates partial destruction of the incipient tumor without concomitant autoimmunity. B) ID8-OVA tumors that are transiently controlled by ACT of OT-3 CD8⁺ T cells demonstrate progressive tumor outgrowth by 21 days post cell transfer.

Fig. 4.. OT-3 CD8 T cells upregulate inhibitory receptors in response to ID8-OVA tumors and become functionally exhausted.

A) 5×10⁶ OT-3 CD8⁺ T cells adoptively transferred to mice bearing mature ID8-OVA tumors and harvested from the tumor draining lymph node 15 days post-transfer are unable to produce IFNγ in response OVA257-264 (SIINFEKL) peptide restimulation, whereas OT-3 CD8⁺ T cells primed by an ID8 tumor cell vaccine that lacks persistence of the tumor cells in vivo demonstrate successfully SIINFEKL antigen-specific restimulation. B) OT-3 CD8⁺ T cells adoptively transferred to mice bearing ID8-OVA tumors and harvested from the tumor draining lymph node 15 days post-transfer demonstrate upregulation of the inhibitory coreceptors PD-1, Tim-3, and Lag-3, whereas mice bearing ID8-WT tumors or mice vaccinated with irradiated ID8-OVA cells did not upregulate exhaustion markers.

Fig. 5.. Cotransfer of OT-3 CD8⁺ T cells with the immune checkpoint inhibitors anti-PD1, anti-TIM3, and anti-LAG3 elicits autoimmune diabetes that correlates with antitumor responses.

A) Intravenous co-administration of the immune checkpoint inhibitors anti-PD1, anti-TIM3, and anti-LAG3 (250 µg) with 5×10⁶ OT-3 CD8⁺ T cells into RIP-mOVA mice bearing 3-week old ID8-OVA tumors did not demonstrate statistically significant changes in tumor killing by tumor bioluminescence by day 7 post-ACT as single agents or in combination vs. mice that only received OT-3 CD8⁺ T cells B) Linear regression analysis of all treatment conditions did demonstrate that tumor killing correlates with blood glucose concentration in treated mice.

Fig. 6.. Combinatorial immune checkpoint blockade with anti-PD1, anti-TIM3, and anti-LAG3 reverses OT-3 CD8⁺ T cell exhaustion with a trend to enhanced tumor killing but induces autoimmune diabetes.

A) Intravenous co-administration of anti-PD1, anti-TIM3, and anti-LAG3 (250 µg) with 5×10⁶ OT-3 CD8⁺ T cells into RIP-mOVA mice bearing 3-week old ID8-OVA tumors induced diabetes in half of the treated mice. B) There is a non-statistically significant trend towards greater tumor killing in mice that developed autoimmune diabetes. SIINFEKL restimulation of splenocytes harvested from treated mice showed higher responses in the diabetic mice vs. non-diabetic mice in this group.